Fatal toxic epidermal necrolysis associated with sinomenine in a patient with primary membranous nephropathy.

Sinomenine (SIN), the alkaloid monomer extracted from Sinomenium acutum, is a kind of non-steroidal anti-inflammatory drug widely used in China to treat rheumatoid arthritis (RA) and various glomerular diseases. It has various pharmacological effects such as anti-inflammatory, analgesic, and anti-tumor. As a strong histamine-releasing agent, SIN has drawn increasing attention in regards to its side effects such as allergic, gastrointestinal, and circulatory systemic reactions. In this report, we first described a patient with primary membranous nephropathy (PMN) who was treated with oral intake of SIN and developed medicine-induced toxic epidermal necrolysis (TEN) and subsequently died of septic multi-organ failure. The present case report intends to demonstrate the underestimated side effects of SIN that can eventually lead to death.

Human-derived decellularized extracellular matrix scaffold incorporating autologous bone marrow stem cells from patients with congenital heart disease for cardiac tissue engineering.

BACKGROUND: Stem cells are used as an alternative treatment option for patients with congenital heart disease (CHD) due to their regenerative potential, but they are subject to low retention rate in the injured myocardium. Also, the diseased microenvironment in the injured myocardium may not provide healthy cues for optimal stem cell function. OBJECTIVE: In this study, we prepared a novel human-derived cardiac scaffold to improve the functional behaviors of stem cells. METHODS: Decellularized extracellular matrix (ECM) scaffolds were fabricated by removing cells of human-derived cardiac appendage tissues. Then, bone marrow c-kit+ progenitor cells from patients with congenital heart disease were seeded on the cardiac ECM scaffolds. Cell adhesion, survival, proliferation and cardiac differentiation on human cardiac decellularized ECM scaffold were evaluated in vitro. Label-free mass spectrometry was applied to analyze cardiac ECM proteins regulating cell behaviors. RESULTS: It was shown that cardiac ECM scaffolds promoted stem cell adhesion and proliferation. Importantly, bone marrow c-kit+ progenitor cells cultured on cardiac ECM scaffold for 14 days differentiated into cardiomyocyte-like cells without supplement with any inducible factors, as confirmed by the increased protein level of Gata4 and upregulated gene levels of Gata4, Nkx2.5, and cTnT. Proteomic analysis showed the proteins in cardiac ECM functioned in multiple biological activities, including regulation of cell proliferation, regulation of cell differentiation, and cardiovascular system development. CONCLUSION: The human-derived cardiac scaffold constructed in this study may help repair the damaged myocardium and hold great potential for tissue engineering application in pediatric patients with CHD.

Whole Blood Transcriptome Analysis Reveals the Correlation between Specific Immune Cells and Septicemic Melioidosis.

Melioidosis is a serious infectious disease caused by the environmental Gram-negative bacillus Burkholderia pseudomallei. It has been shown that the host immune system, mainly comprising various types of immune cells, fights against the disease. The present study was to specify correlation between septicemic melioidosis and the levels of multiple immune cells. First, the genes with differential expression patterns between patients with septicemic melioidosis (B. pseudomallei) and health donors (control/healthy) were identified. These genes being related to cytokine binding, cell adhesion molecule binding, and MHC relevant proteins may influence immune response. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed 23 enriched immune response pathways. We further leveraged the microarray data to investigate the relationship between immune response and septicemic melioidosis, using the CIBERSORT analysis. Comparison of the percentages of 22 immune cell types in B. pseudomallei vs. control/healthy revealed that those of CD4 memory resting cells, CD8+ T cells, B memory cells, and CD4 memory activated cells were low, whereas those of M0 macrophages, neutrophils, and gamma delta T cells were high. The multivariate logistic regression analysis further revealed that CD8+ T cells, M0 macrophages, neutrophils, and naive CD4+ cells were strongly associated with the onset of septicemic melioidosis, and M2 macrophages and neutrophils were associated with the survival in septicemic melioidosis. Taken together, these data point to a complex role of immune cells on the development and progression of melioidosis.

A CTLA-4 blocking strategy based on Nanoboby in dendritic cell-stimulated cytokine-induced killer cells enhances their anti-tumor effects.

BACKGROUND: Cytokine-induced killer cells induced with tumor antigen-pulsed dendritic cells (DC-CIK) immunotherapy is a promising strategy for the treatment of malignant tumors. However, itsefficacy isrestricted by the immunosuppression, which is mediated by the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) pathway. In order to overcome the negative co-stimulation from these T cells,we screened a nanobody targeted for CTLA-4 (Nb36) and blocked the CTLA-4 signaling with Nb36. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors to beused to induce CIK cells in vitro, after which they were co-cultured with DC cells that had received tumor antigens. In addition, wetested whether blocking CTLA-4 signaling with Nb36 could promote in vitro DC-CIK cells proliferation, pro-inflammatory cytokine production and cytotoxicity,or not. For the in vivo experiments, we constructed a subcutaneously transplanted tumor model and placed it in NOD/SCID mice to verify the anti-tumor effect of this therapy. RESULTS: After stimulation with Nb36, the DC-CIK cells presented enhanced proliferation and production of IFN-γ in vitro, which strengthened the killing effect on the tumor cells. For the in vivo experiments, it was found that Nb36-treated DC-CIK cells significantly inhibited the growth of subcutaneously transplanted livercancer tumors, as well as reduced the tumor weight and prolonged the survival of tumor-bearing NOD/SCID mice. CONCLUSIONS: Ourfindings demonstrated that in response to CTLA-4 specific nanobody stimulation, DC-CIK cells exhibited a better anti-tumor effect. In fact, this Nb-based CTLA-4 blocking strategy achieved an anti-tumor efficacy close to that of monoclonal antibodies. Our findings suggest that DC-CIK cells + Nb36 have the potential totreatmalignant tumors through in vivo adoptive therapy.

O-GlcNAcylation of core components of the translation initiation machinery regulates protein synthesis.

Protein synthesis is essential for cell growth, proliferation, and survival. Protein synthesis is a tightly regulated process that involves multiple mechanisms. Deregulation of protein synthesis is considered as a key factor in the development and progression of a number of diseases, such as cancer. Here we show that the dynamic modification of proteins by O-linked ß-N-acetyl-glucosamine (O-GlcNAcylation) regulates translation initiation by modifying core initiation factors eIF4A and eIF4G, respectively. Mechanistically, site-specific O-GlcNAcylation of eIF4A on Ser322/323 disrupts the formation of the translation initiation complex by perturbing its interaction with eIF4G. In addition, O-GlcNAcylation inhibits the duplex unwinding activity of eIF4A, leading to impaired protein synthesis, and decreased cell proliferation. In contrast, site-specific O-GlcNAcylation of eIF4G on Ser61 promotes its interaction with poly(A)-binding protein (PABP) and poly(A) mRNA. Depletion of eIF4G O-GlcNAcylation results in inhibition of protein synthesis, cell proliferation, and soft agar colony formation. The differential glycosylation of eIF4A and eIF4G appears to be regulated in the initiation complex to fine-tune protein synthesis. Our study thus expands the current understanding of protein synthesis, and adds another dimension of complexity to translational control of cellular proteins.

Chemical remodeling cell surface glycans for immunotargeting of tumor cells.

Recruitment of human endogenous antibodies to target and eliminate tumor cells is a promising therapeutic strategy in the biomedical field. Current antibody-recruiting molecules are typically bi-functional agents that utilize cell-surface receptor binding property for targeting. This approach has intrinsic limitations due to the heterogeneity of tumor cells and the limited number of receptors on the cell surface. Here we report a targeting strategy based on remodeling of cell surface glycans through metabolic engineering and bioorthogonal chemical ligation. In vitro cultured tumor cells and in vivo xenograft tumors were actively remodeled with rhamnose carbohydrate epitopes, which were capable of recruiting endogenous anti-rhamnose antibodies and activating complement-mediated cell cytotoxicity. This study highlights the therapeutic potential for modulating endogenous immune response through cell-surface glycan engineering.

Imaging of protein-specific glycosylation by glycan metabolic tagging and in situ proximity ligation.

Glycosylation is an important posttranslational modification, which regulates a number of critical biological processes including cell-cell recognition, signal transduction and disease progression. Probing the glycosylation status on a specific protein of interest enables an in-depth understanding of the role of glycosylation on protein structure and function. However, methods for monitoring protein-specific glycosylation are largely lacking. Here we describe a highly sensitive fluorescence imaging strategy to visualize the protein-specific glycosylation by combining glycan metabolic tagging and in situ proximity ligation (termed GPLA). We demonstrate the visualization of sialylation, fucosylation and GalNAcylation on several important membrane proteins. Notably, the high spatial resolution of this method allows subcellular localization of the glycosylated fraction of the proteins. We further show that our strategy can be applied to image the dimerization of endogenous epidermal growth factor receptor. Thus, our study provides a unique tool to monitor the protein-specific glycosylation in a dynamic cellular context.

Targeting Tumor Cells by Natural Anti-Carbohydrate Antibodies Using Rhamnose-Functionalized Liposomes.

Recruitment of antibodies in human immune systems for targeted destruction of tumor cells has emerged as an exciting area of research due to its low occurrence of side effects, high efficacy, and high specificity. The presence of large amounts of anticarbohydrate natural antibodies in human sera has prompted research efforts to utilize carbohydrate epitopes for immune recruitment. Here, we have developed a general strategy for specific targeted destruction of tumor cells based on rhamnose-functionalized liposomes. Tumor cells artificially decorated with rhamnose epitopes were subjected to complement-mediated cytotoxicity in vitro and showed delayed tumor growth in vivo. This study highlights the therapeutic potential for activation of endogenous immune response through cell-surface glycan engineering.

O-GlcNAcylation of G6PD promotes the pentose phosphate pathway and tumor growth.

The pentose phosphate pathway (PPP) plays a critical role in macromolecule biosynthesis and maintaining cellular redox homoeostasis in rapidly proliferating cells. Upregulation of the PPP has been shown in several types of cancer. However, how the PPP is regulated to confer a selective growth advantage on cancer cells is not well understood. Here we show that glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, is dynamically modified with an O-linked ß-N-acetylglucosamine sugar in response to hypoxia. Glycosylation activates G6PD activity and increases glucose flux through the PPP, thereby providing precursors for nucleotide and lipid biosynthesis, and reducing equivalents for antioxidant defense. Blocking glycosylation of G6PD reduces cancer cell proliferation in vitro and impairs tumor growth in vivo. Importantly, G6PD glycosylation is increased in human lung cancers. Our findings reveal a mechanistic understanding of how O-glycosylation directly regulates the PPP to confer a selective growth advantage to tumours.

MAPKs are not involved in triptolide-induced cell growth inhibition and apoptosis in prostate cancer cell lines with different p53 status.

Triptolide showed excellent antitumor activity against several solid tumors. However, its mechanism has not been fully understood. To further elucidate it, the effects of mitogen activated protein kinases (MAPKs) on the activity of triptolide towards prostate cancer cell lines were investigated in the present study using both LNCaP (p53 positive and androgen-dependent) and PC-3 (p53 deficient and androgen-independent) cells. Our results showed that triptolide exerted potent growth inhibitory and apoptotic effects on both cell lines, and the effects were independent of the expression of p53. Although upregulation of ERK and JNK phosphorylation was observed after the triptolide treatment, the results with inhibitors showed that these MAPKs were not involved in the mechanism of triptolide activity in human prostate cancer cell lines with different p53 status.

[Soluble-expression, purification and activity analysis of extracellular domain III of flt1].

To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.